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Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.

ABSTRACT: RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPgammaS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 A x 130 A with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.

SUBMITTER: Muzzolini L

PROVIDER: S-EPMC1769423 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.

Muzzolini Laura L Beuron Fabienne F Patwardhan Ardan A Popuri Venkateswarlu V Cui Sheng S Niccolini Benedetta B Rappas Mathieu M Freemont Paul S PS Vindigni Alessandro A

PLoS biology 20070201 2

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and tran ...[more]

PMID: 17227144

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Project description:BackgroundNon-histone protein acylation is increasingly recognized as an important posttranslational modification, but little is known as to the biochemical properties of protein serine acylating enzymes.Methodology/principal findingsWe here report that we have identified a metal-stimulated serine octanoyltransferase activity in microsomes from human erythroleukemic (HEL) cells. The HEL acylating enzyme was linear with respect to time and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc), and inhibited by chelating reagents. Hydroxylamine treatment removed most, but not all, of the attached radioactivity. A salt extract of microsomal membranes contained the major portion of enzyme activity, indicating that this acyltransferase is not an integral membrane protein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competition experiments, the acyltransferase was well inhibited by activated forms of fatty acids containing at least eight to fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptides des-acyl ghrelin and ACTH were also not acylated; however, des-acyl ghrelin containing the N-terminal tripeptide Gly-Ala-Met was acylated. Mutagenesis studies indicated a requirement for serine five residues from the amino terminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, properties preserved in the HEL cell enzyme purified through four sequential chromatographic steps.Conclusions/significanceIn conclusion, our data demonstrate the presence of a zinc-stimulated acyltransferase activity concentrated in the endoplasmic reticulum in HEL cells which is likely to contribute to medium-chain protein lipidation.

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Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.

Publications

Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.

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